Production of hyaluronic acid

ABSTRACT

A process for the production of hyaluronic acid by continuous fermentation of Streptococcus equi in a chemostat culture gives high yields of high molecular weight hyaluronic acid uncontaminated by toxic impurities. The process is advantageous in that it solves the problem of traditional batch culture in which degradation enzymes can begin to break down the cell walls of Streptococcus releasing cell contents into the fermenter broth, leading to purification difficulties.

The present invention relates to a process for the production ofhyaluronic acid (HA) by bacterial fermentation.

HA is a member of a class of polymers known as glycosaminoglycans. HA isa long chain linear polysaccharide and is usually present as the sodiumsalt which has a molecular formula of (C₁₄ H₂₀ NNaO₁₁)n where n can varyaccording to the source, isolation procedure and method ofdetermination. However, molecular weights of up to 14×10⁶ have beenreported.

HA and its salts can be isolated from many sources including nearly allconnective matrices of vertebrate organisms. However, HA is also acapsular component of bacteria such as Streptococci as was shown byKendall et al, (1937), Biochem. Biophys. Acta, 279, 401-405.

HA is non-immunogenic and therefore has great potential in medicine. HAhaving a high molecular weight (over 1 million) has been found to beparticularly useful because of its visco-elastic properties. The HAwhich is at present commercially available is generally obtained fromavian sources such as rooster combs but problems with this materialinclude the likelihood of it being contaminated by viruses. Complexpurification procedures are therefore needed and a suitable process isdescribed in U.S. Pat. No. 4,141,973. However, the need for thisextensive purification clearly adds to the production cost of thematerial.

Because of the problems associated with the isolation of HA from aviansources, attempts have been made to develop fermentation processes inwhich HA is produced. Although all species of Streptococcus produce HA,it is important to choose a species which is a good producer of HA andwhich is free of hyaluronidase activity.

U.S. Pat. No. 4,517,295 describes a fermentation process using S.pyogenes but the product has an average molecular weight of only 55,000.EP-A-0144019 describes an alternative fermentation process using S. equiwhich claims to produce a high molecular weight HA but the molecularweight is calculated by a non-standard method and cannot thereforeeasily be compared with molecular weights calculated by other methods.WO-A-8604355 and U.S. Pat. No. 4,897,349 both describe fermentationprocesses in which HA of high molecular weight is produced in good yieldbut in both of the processes, a pathogenic species of Streptococcus isused and so the HA product is likely to be unsuitable for use inmedicine because of contamination by the bacterial toxins.

In addition, all of the prior art processes described are batchfermentation processes. There are various problems with batchfermentation processes and these include production of a contaminatedproduct which is difficult to purify.

It would therefore be particularly advantageous to develop afermentation process which is free from the usual disadvantages of batchfermentation and in which HA having a high molecular weight (for exampleseveral million) could be produced.

In a first aspect of the invention, therefore, there is provided aprocess for the production of HA by fermentation of Streptococcus,characterised in that the process comprises continuous fermentation ofStreptococcus in a chemostat culture which is maintained at a pH of from6.0 to 7.0, a dilution rate of 0.05 to 0.12 h⁻¹ and a dissolved oxygentension of less than 1% saturation.

Continuous fermentation processes are known and the theory has beendescribed by Herbert et al (1956) J. Gen. Micro., 14, 602-622. Thenumber of commercial continuous fermentation processes are limitedbecause of the perceived difficulty of continuous fermentation processesover traditionally based batch processes. Also, continuous fermentationprocesses have traditionally been considered to be suited only for largeproduction output, low product value facilities whereas batch culturehas always been used for low production output high product valuefacilities such as those used to make HA.

The process of the invention overcomes various problems associated withtraditional batch culture technique. In batch culture, as theStreptococcus approaches stationary phase, various degradation enzymesstart to break down the cells releasing cell contents into the fermenterbroth and this leads to purification difficulties. This does not occurif a continuous fermentation process is used since the fermentationmedium is maintained in a steady state so that the expression of suchenzymes is reduced. A further advantage of the steady state obtainedwith continuous fermentation is that the cell wall turnover is reducedwhich is advantageous because it has proved extremely difficult toseparate HA from cell wall components which have been released into thefermentation medium. Finally, the use of continuous culture avoids theexpression of various toxins which are expressed during the stationaryphase of batch cultures. The use of a continuous fermentation processtherefore allows for the production of a much purer product.

The HA produced by the process of the invention has an average molecularweight of from 1 to 3 million and under preferred conditions, themolecular weight is from 1.6 to 2.5 million. High molecular weight HA insolution has visco-elastic properties which make it extremely useful ina variety of clinical fields including wound treatment, ophthalmicsurgery and orthopaedic surgery. HA is also potentially useful in avariety of non-medical fields.

If the HA produced by the process of the invention is to be useful inmedicine, it is of course important that any contaminants should not betoxic. The species of Streptococcus used in the fermentation processshould therefore preferably be one which is not a human pathogen inorder to minimise the risk that any bacterial contaminants remaining inthe product will then be toxic. In addition, the safety of themanufacturing facility is increased if a non pathogenic Streptococcus isused and an accidental leak from a fermenter will not cause serioushealth risks. A particularly suitable species for use in thisfermentation process is S. equi although other species could of coursebe used.

Suitable strains of S. equi for use in the process will easily beselected by those skilled in the art using long term selection in thechemostat culture and choosing a stable, high yielding phenotypicvariant suitable for long term culture by isolating from the culture,individual cells to use as seed for further fermentations. A sample ofthe culture may be streaked on to solid medium and colonies originatingfrom individual (or a small number of) cells allowed to grow. We havefound that the starting strain of S. equi may be improved by selectingfast growing colonies with large mucoid capsules having a stringyappearance when pulled with a loop. These colonies may be used to seedthe fermenter for the next run. Preferably, they are first subculturedonto further plates and the same selection criteria applied to selectseed-colonies.

A particularly suitable strain has been deposited by us under theBudapest Treaty at the National Collection of Industrial and MarineBacteria (NCIMB), 23 St. Machai Drive, Aberdeen, Scotland AB2 IRY on 24Oct. 1990 under the accession No NCIMB 40327.

The fermentation process of the invention takes place in a nutrientmedium containing the following components:

an assimilable source of carbon;

a source of nitrogen;

sources of phosphorus, sodium, potassium, magnesium, iron, zinc andmanganese;

sources of growth factors; and

a source of sulphur.

Carbon may be supplied in the form of a sugar, particularly glucose,although sucrose can also be used. The source of nitrogen may be anon-toxic nitrogen containing salt, particularly a water soluble salt,for example, an ammonium salt such as ammonium chloride. The metals andphosphorus may also be supplied in the form of water soluble salts. Thenecessary growth factors are all contained in a source such as yeastextract which may also be the source of the sulphur which is required.

The nutrient medium may additionally contain sources of one or more ofcalcium, molybdenum, cobalt copper or boron.

The growth rate of the bacteria in the continuous fermentation processmay be controlled by limiting the availability of one essentialcomponent of the nutrient medium, thus limiting biomass production butnot energy conversion or polysaccharide formation. The supply of any ofthe essential components listed above may be limited but it is preferredto limit the supply of the sulphur.

The pH of the nutrient medium must, as mentioned above, be maintainedwithin the range of 6.0 to 7.0. A preferred range is 6.0 to 6.4 and mostfavourable conditions are achieved when the pH is 6.2.

The pH of the medium may be maintained within the desired range by theaddition of an alkali such as sodium hydroxide during the fermentationprocess. Any foaming which results may be controlled by the addition ofa suitable non-toxic foaming agent, for example an agent based onpolypropyleneglycol.

As discussed above, the nutrient medium is supplied to the fermentationzone at a dilution rate (flow rate per unit volume of the fermenter) offrom 0.05 to 0.12 h⁻¹. The most favourable conditions occur when thedilution rate is about 0.07 h⁻¹. Effluent is withdrawn from thefermenter at a rate equal to the rate of supply of nutrient medium tothe fermenter so as to maintain a constant volume of medium within thefermentation vessel. A constant physicochemical environment ismaintained using automatic controllers which maintain constant optimumconditions for the selected strain of S. equi during long termcontinuous culture. The product is harvested from the effluent.

The culture of Streptococcus is carried out under microaerophilicconditions. Air or other oxygen containing gas may be pumped into theculture medium at a rate sufficient to maintain a dissolved oxygentension in the fermentation medium of less than 1% saturation andpreferably in the range of 0.1 to 0.5% saturation. The gas supplied tothe fermentation medium must be sterile and, in the case where air isused, a suitable flow rate may be from 0.1 to 0.5 v.v.m. (volumes perfermenter volume per minute).

It is desirable that the fermentation is carried out with continuousagitation since the fermentation medium is viscous and thorough mixingof the medium is essential to eliminate "dead zones" in which growth ofundesired non-HA producing strains of S. equi can occur.

The temperature of the fermentation medium should be maintained in therange of 30° to 40° C. but a preferable range is from 35° to 40° C. andthe most suitable temperature is 37° C.

Under the preferred operating conditions, the process of the inventionhas yielded as much as 2.5 g of HA for every litre of fermentationmedium and it is possible that even higher yields may be obtained.Stable culture conditions can be maintained for over 500 hours. Theprocess therefore represents a considerable improvement over prior artbatch fermentation processes.

After the fermentation process, the biomass is killed and the HAextracted with an aqueous medium containing an anionic surfactant. Thesetwo steps may take place either simultaneously or sequentially. Avariety of means may be used to kill the biomass, including heat or akilling agent such as an antibacterial substance. A particularlysuitable agent for killing the biomass is formaldehyde which may be usedas the aqueous solution commonly known as formalin. A suitableconcentration of the solution is from 0.5 to 1.5% (v/v). The surfactantmay be added at a concentration of from 0.01 to 0.05% (w/v) andpreferably at a concentration of 0.02% (w/v). A suitable surfactant forextracting the HA from the killed biomass is sodium dodecyl sulphate.

The biomass is kept in contact with the killing agent and the surfactantuntil substantially all the HA has been released from the cellularcapsules and this may take from 10 to 24 hours, usually about 16 hours.

The residual biomass is then separated from the aqueous solution byfiltration, for example using a plate and frame filter process and anappropriate filtration medium such as kieselguhr filter pads. It isnecessary to clean or replace these filter pads during the fermentationprocess and a suitable time interval for this may be every 24 hours. Analternative filtration method which is particularly useful for largescale operation is to use a filter cartridge. Such cartridges have to bereplaced at similar time intervals to the filter pads. After thefiltration, a cell-free filtered solution of HA is obtained and thissolution may be purified by diafiltration to remove low molecular weightimpurities. These impurities are those derived from the productionorganism's metabolism, the residual components of the nutrient medium,residual killing agent and residual anionic surfactants. It is necessaryin this step to use an ultra filtration membrane with an appropriatemolecular weight cut off which is usually from 10,000 to 25,000 Daltons,and preferably 20,000 Daltons nominal molecular weight. Suitablemembranes are based on polysulphones and are available commercially. Thefiltered solution containing the dissolved HA is diafiltered againstfrom 8 to 20 volumes, preferably about 10 volumes of purified water andthe liltrate is continuously discarded. Water of suitable purity has aconductivity of less than 10 μScm⁻¹.

After the diafiltration, the molarity of purified solution of HA isadjusted to within the range of 0.18 to 0.24M, preferably 0.20M withrespect to sodium chloride. The pH is also adjusted if necessary to avalue of from 6.3 to 7.8, preferably from 7.0 to 7.5. The mostfavourable results are achieved when the pH is 7.2. The pH may beadjusted by the addition of a base such as sodium hydroxide or anappropriate buffer, particularly a phosphate buffer.

If a product of medical grade is required, the process may include anoptional step of precipitating nucleic acids from the solution. This isachieved by the addition of a cationic surfactant for example aquaternary ammonium compound such as cetyl pyridinium chloride. Thecationic surfactant may be added as a dilute aqueous solution; forexample a 1% (w/v) solution of cetyl pyridinum chloride may be added ina volume ratio of 1:60 to the solution. The precipitated nucleic acidmay then be removed by filtration through an appropriate filter mediumranging in pore size of from 3.2 μm to 0.2 μm but preferably from 1.2 μmto 0.2 μm.

If this step is used, subsequent processing must be carried out understerile conditions using pyrogen-free equipment.

After this optional stage, or, if the optional stage is not used, afterthe adjustment of the molarity and pH of the solution, HA isprecipitated by the addition of a non-solvent, for example a loweralcohol such as isopropyl alcohol. The precipitated HA is filtered offand the filtrate discarded.

Further purification of the HA product can be achieved by redissolvingthe HA in sodium chloride solution and then reprecipitating by additionof a non-solvent in the same way as described above. The sodium chloridesolution must have a molarity in the range of 0.18 to 0.24M, the mostfavourable value being 0.20M. The pH of the solution must be from 6.3 to7.8 but is preferably 7.0 to 7.5 and most preferably 7.2. The solutionmay be buffered, for example with a phosphate buffer.

In a second aspect of the invention there is provided hyaluronic acidproduced by the process of the first aspect. This HA has an averagemolecular weight of at least 1 million and the range of molecularweights of the product is preferably from 1.6 to 2.5 million.

In a third aspect of the invention, there is provided Streptococcus equiof the strain NCIMB 40327.

The invention will now be further described with reference to thefollowing examples.

EXAMPLE 1

A growth medium for S. equi is formulated as follows:

    ______________________________________                                        Glucose             60.00   g                                                 Yeast extract       6.25    g (Oxoid L21)                                     Sodium dihydrogen phosphate (2H.sub.2 O)                                                          2.02    g                                                 Ammonium chloride   2.14    g                                                 Potassium chloride  0.71    g                                                 Citric acid         0.42    g                                                 Magnesium oxide     0.40    g                                                 Calcium carbonate   0.10    g                                                 Sodium molybdate (2H.sub.2 O)                                                                     2.42    mg                                                Ferrous chloride (6H.sub.2 O)                                                                     10.80   mg                                                Cobalt chloride (6H.sub.2 O)                                                                      0.95    mg                                                Copper chloride (2H.sub.2 O)                                                                      0.32    mg                                                Zinc oxide          0.81    mg                                                Manganese chloride (6H.sub.2 O)                                                                   4.00    mg                                                Boric acid          0.12    mg                                                Conc. Hydrochloric acid                                                                           0.178   mL                                                ______________________________________                                    

This medium is made up to 1 L with purified water. It is then sterilizedby filtration through an 0.22 μm absolute rated filter.

The fermentation medium is pumped continuously into the fermenter at aflow rate, in relation to the fermenter volume, of 0.07 h⁻¹. The mediumis aerated with sterile air which has been filtered through an 0.2 μmabsolute rated filter. The air flow rate is maintained at 0.2 v.v.m. anda dissolved oxygen .tension is maintained in the fermenter broth at 0.2%saturation. Streptococcus equi is grown in this culture medium at 37° C.The pH is maintained at 6.2 by automatically controlled additions ofsodium hydroxide. Foam generation is controlled by addition as necessaryof a polypropylene glycol based antifoam.

Fermentation medium is continuously withdrawn from the fermenter at thesame rate as the fresh medium is fed in. This effluent contains about2.5 gL⁻¹ of hyaluronic acid. Sodium dodecyl sulphate and formalinsolution are continuously fed into the effluent from the fermenter toachieve final concentrations of 0.025% (w/v) of sodium dodecyl sulphateand 1% (v/v) of formalin. Mixing of the effluent and the sodium dodecylsulphate/formalin streams takes place in an in-line static mixer. Thecontact time is 16 hours and, after mixing, the stream passes through avessel designed for this residence time.

After release of the hyaluronic acid into the aqueous medium, theresidual biomass is removed continuously by depth filtration in acartridge filter using filters of appropriate pore size. Duplicatefilter units are used to allow periodic diversion of the product flow toa clean filter, thus allowing cleaning and replacement of used filters.The filter units are sized to allow up to 24 hours operation before flowdiversion is required.

The solution from which the cells have been removed is then processed bydiafiltration against purified water to remove residual materials fromthe culture medium, sodium dodecyl sulphate and formalin. Thisdiafiltration is operated using a polysulphone based ultrafiltrationmembrane with a 20,000 Dalton nominal molecular weight cut off. Thesolution is diafiltered against ten volumes of water having aconductivity of less than 10 μS cm⁻¹, and the filtrate is continuouslydiscarded. After diafiltration, sodium chloride (final concentration0.2M) is added to the solution obtained and the pH adjusted to 7.2 byaddition of phosphate buffer (Na₂ HPO₄, 0.22 gL⁻¹ ; NaH₂ PO₄.2H₂ O,0.045 gL⁻¹). A 1% (w/v) solution of cetyl pyridinium chloride is thenadded in a ratio of about 1:60 by volume. The nucleic acids thusprecipitated are removed by pumping the solution through 1.2 μm and 0.2μm (absolute rated) depth filters arranged in series. The filteredsolution of hyaluronic acid thus obtained is then continuously mixed inline with a metered flow of isopropyl alcohol at a flow ratio of 1:.2.The mixing is performed in a static mixer and the precipitatedhyaluronic acid is separated from the aqueous solution in a basketfilter. The filtrate is discarded. The recovered hyaluronic acid isredissolved in 0.2M sodium chloride solution buffered at pH 7.2 withphosphate (Na₂ HPO₄, 0.022 gL⁻¹ ; NaH₂ PO₄.2H₂ O, 0.045 gL⁻¹) to give anHA concentration of 0.2% w/v). The hyaluronic acid is again precipitatedfrom this solution by addition of isopropyl alcohol in the same way aspreviously.

The precipitated hyaluronic acid is washed with isopropyl alcohol andthe washings are discarded. Final traces of isopropyl alcohol areremoved by drying in air under sterile conditions. All the purificationprocedures are carried out at ambient temperature.

A medical grade solution may be made by dissolving hyaluronic acidproduced in the manner described in 0.15M sterile saline solutionbuffered with the phosphate buffer mentioned above, pH 7.3 to give a 1%(w/v) solution of hyaluronic acid. The sodium hyaluronate solution soprepared has an average molecular weight of 1.6 to 2.5×10⁶ Da asdetermined by low angle laser light scattering techniques andviscometry. The solution has a protein content of less than 0.2% (w/w)and a nucleotide level of less than 0.15% (w/w). The 1% (w/v) solutionshows a U.V. absorption of 0.14 AU at 260 nm and 0.1 AU at 280 nm. Theviscosity of the solution is 159 Pa.s at zero shear falling to less than1 Pa.s at 1000 s⁻¹.

The following example demonstrates a method of selecting suitablestrains of S. equi for use in the fermentation process.

EXAMPLE 2

A solid medium for growth of S. equi is formulated as follows:

    ______________________________________                                        Glucose            20       g                                                 Yeast extract      5        g (Oxoid L21)                                     Agar               15       g (Oxoid L 13                                                                 .sup.  Agar No. 3)                                Di-potassium hydrogen                                                                            1.706    g                                                 orthophosphate anhydrous                                                      Potassium dihydrogen ortho-                                                                      1.388    g                                                 phosphate                                                                     Sodium dihydrogen orthophosphate                                                                 2.92     g                                                 Ammonium chloride  5.01     g                                                 Potassium chloride 372      mg                                                Citric acid        420      mg                                                Magnesium oxide    50.4     mg                                                Calcium carbonate  10       mg                                                Sodium molybdate   2.4      mg                                                Ferrous chloride (6H.sub.2 O)                                                                    270      mg                                                Cobalt chloride (6H.sub.2 O)                                                                     2.37     mg                                                Copper chloride (2H.sub.2 O)                                                                     0.85     mg                                                Zinc oxide         2.05     mg                                                Manganese chloride (4H.sub.2 O)                                                                  10       mg                                                Boric acid         0.3      mg                                                Conc. Hydrochloric acid                                                                          0.24     mL                                                ______________________________________                                    

The glucose is dissolved in 50 mL of water, the yeast extract in 100 mLand the agar/salts dissolved in 820 mL of water, sterilized separatelyby autoclaving at 121° C. for 15 minutes and then mixed together priorto pouring the plates.

Samples of S. equi from a fermentation experiment performed as describedin Example 1 were obtained for strain improvement. Strain improvementwas carried out by selecting large fast growing domed colonies withlarge mucoid capsules having a stringy appearance when pulled with aloop. These colonies were subcultured onto further plates where the sameselection criteria were applied.

It can therefore be seen that the process of the invention provides aneconomically viable fermentation process for obtaining pure HA of highmolecular weight and therefore represents a significant improvement overprior art processes.

We claim:
 1. A process for the production of hyaluronic acidcomprising:(a) culturing Streptococcus equi by fermentation in anutrient medium containing:an assimilable source of carbon; a source ofnitrogen; sources of phosphorus, sodium, potassium, magnesium, iron,zinc and manganese; sources of growth factors; and a source ofsulphur,characterized in that the process comprises continuousfermentation of Streptococcus equi in a chemostat culture which ismaintained at a pH of from 6.0 to 7.0, a dilution rate of 0.05 to 0.12h⁻¹, and a dissolved oxygen tension of less than 1% saturation toproduce a biomass containing hyaluronic acid, and (b) recovering saidhyaluronic acid from said biomass.
 2. The process of claim 1, whereinsaid source of carbon in the nutrient medium is a sugar.
 3. The processof claim 1, wherein said source of nitrogen in the nutrient medium is anammonium salt.
 4. The process of claim 1, wherein said sources ofphosphorus, sodium, potassium, magnesium, iron, zinc and manganese inthe nutrient medium are water soluble salts of those elements.
 5. Theprocess of claim 1, wherein said source of growth factors and sulphur inthe nutrient medium is yeast extract.
 6. The process of claim 1, whereinsaid nutrient medium further comprises sources of one or more membersselected from the group consisting of calcium, molybdenum, cobalt,copper and boron.
 7. The process of claim 1, wherein said sulphur is alimiting component.
 8. The process of claim 1, wherein said nutrient.medium contains limited amounts of one or more members selected fromthe group consisting of carbon, nitrogen, phosphorous, sodium,potassium, magnesium, iron, zinc, manganese, growth factors and sulphur.9. The process of claim 1, wherein said pH of said nutrient medium is6.2.
 10. The process of claim 1, wherein said dilution rate is 0.07 h⁻¹.11. The process of claim 1, wherein said dissolved oxygen tension insaid chemostat culture is in the range of 0.1 to 0.5% saturation. 12.The process of claim 1, wherein said continuous fermentation is carriedout with continuous agitation.
 13. The process of claim 1, wherein thetemperature is maintained in the range of 35° to 40° C.
 14. The processof claim 1, wherein said Streptococcus is Streptococcus equi NCIMB40327.